Pichia pastoris is a methylotrophic yeast. It was discovered in the 1960s and is characterized by its ability to use methanol as its sole carbon and energy source. After years of research, Pichia pastoris has been widely used in biochemical research and the biotechnology industry, gradually becoming an important production system for recombinant protein expression. Although the application history and reputation of Pichia pastoris are far less than that of saccharomyces cerevisiae, which was discovered by humans thousands of years ago, the superior performance of Pichia pastoris compared to saccharomyces cerevisiae is causing its application in biopharmaceuticals to gradually surpass that of saccharomyces cerevisiae. The following table is a partial list of FDA-approved biopharmaceuticals produced in yeast cells, including hormones, vaccines, blood-related products, cytokines, and enzymes, etc.
FDA-approved biopharmaceuticals produced in yeast cells (adapted from Walsh, 2018).[1]
Initially, the biopharmaceutical industry had high hopes for saccharomyces cerevisiae, expecting this eukaryotic microorganism to have recombinant protein yields and production costs comparable to E. coli, while also possessing eukaryotic cell systems such as post-translational modifications like glycosylation, better folding, and secretion systems. However, "the ideal is rich, but reality is stark." The application of saccharomyces cerevisiae expression system in pharmaceuticals was not smooth at first, and it cast a shadow over the development of yeast in biopharmaceuticals for a time, to the extent that the application of yeast in biopharmaceuticals only began to slowly take off in the 1980s.
The initial difficulties in using yeast recombinant expression in biopharmaceuticals were mainly due to the following issues. The first few recombinant proteins expressed using saccharomyces cerevisiae yielded unsatisfactory results, and the scale-up technology transfer from shake flask to fermentation processes was also mostly unsatisfactory. Additionally, the N-linked glycosylation of saccharomyces cerevisiae differs greatly from that of mammalian cells. Yeast introduces mannose into glycosylation, which can reach lengths of 50-150 units, with great variability or heterogeneity in length, and the core oligosaccharides of the sugar chains contain α-1,3 glycosidic bonds. Both the mannose side chains and α-1,3 glycosidic bond sugar chains can trigger immune responses in the human body, leading to significantly shortened serum half-lives of the products. More importantly, this poses a huge potential risk to the biological safety of the products.
Major N-glycosylation pathways in humans and yeast.[2]
However, with the rapid breakthroughs and developments in molecular biology techniques since the 1970s, people naturally thought of optimizing the glycosylation types and methods of yeast through genetic modification, humanizing the glycosylation process, which is also a direction for optimizing yeast recombinant expression systems.
At the same time, people turned their attention to new yeast strains. At this point, Pichia pastoris emerged as a standout due to its numerous advantages as a biological expression system.
1. Compared to saccharomyces cerevisiae, Pichia pastoris has advantages in the glycosylation of secreted proteins. Unlike Pichia pastoris, N-linked glycosylation in saccharomyces cerevisiae is mainly in the form of high mannose. Saccharomyces cerevisiae typically adds 50-150 mannose residues to oligosaccharide side chains, resulting in large length variations and high heterogeneity of modified proteins. In contrast, Pichia pastoris averages 8-14 mannose residues per side chain, with less length variation, resulting in higher consistency of modified proteins. Moreover, Pichia pastoris does not produce immunogenic α-1,3 glycosidic bonds, making its expressed biopharmaceuticals safer.
2. Compared to saccharomyces cerevisiae, Pichia pastoris has significant advantages in expression regulation. As a methylotrophic yeast, Pichia pastoris can use the AOX1 promoter to express foreign proteins. This is a highly controlled, high-expression induction system without expression leakage issues. Its expression levels can be 10-100 times higher than that of saccharomyces cerevisiae and can even be comparable to E. coli expression systems. It also contains the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level constitutive expression. While saccharomyces cerevisiae expression vectors are in the form of plasmids, Pichia pastoris is integrated into the genome, resulting in higher expression stability and reliability.
3. Compared to expression systems of other species, Pichia pastoris's advantages are also evident, combining the benefits of both prokaryotic and eukaryotic expression systems. Compared to the most commonly used E. coli and CHO expression systems, the specific comparisons are as follows:
Basic characteristics of different host systems for the expression of recombinant proteins.[3]
As can be seen from the above table, although E. coli and CHO expression systems have some advantages in individual comparisons, overall, Pichia pastoris possesses the advantages of simple and economical cultivation of microorganisms, support for high-density fermentation, and high expression levels. It also has the advantages of mammalian cells in post-translational protein modification, correct folding, and secretory expression. In summary, the advantages of Pichia pastoris include simple and economical cultivation, capability for folding and post-translational modifications, high expression yields, and products free from endotoxin and viral contamination.
4. Similar to the genomic integration expression of CHO cell expression platforms, Pichia pastoris can also apply pressure selection for
screening multi-copy recombinants and strain seed bank creation.
However, Pichia pastoris uses single-crossover tandem recombination at a single
fixed site on the yeast chromosome, resulting in better genetic stability of
production cell lines.
So what are the commonly used strains of Pichia pastoris? Next, we will classify them according to mutation types. Pichia pastoris contains two alcohol oxidase genes, AOX1 and AOX2, which enable it to use methanol as a nutrient and energy source. However, the AOX1 product has higher enzyme activity and plays the main role, while the AOX2 product has lower enzyme activity. When AOX1 is mutated, Pichia pastoris can still grow in methanol-containing media, but at a much slower rate. Therefore, based on different AOX1 and AOX2 genotypes, yeast strains can be classified into Mut+ (containing both AOX1 and AOX2), MutS (AOX1 knocked out, AOX2 retained), and Mut- (both AOX1 and AOX2 knocked out). The Mut classification of commonly used Pichia pastoris strains is as follows:
Pichia pastoris can express both secreted proteins and
intracellular proteins, and is widely used in the expression and preparation of
subunit vaccines. Recent progress is shown in the three tables below, which
describe information about expression vectors, marker genes, expression
strains, and target proteins.
Common Pichia pastoris expression vectors for the production of secretory proteins.[3]
Common Pichia pastoris expression vectors for the production of intracellular proteins.[3]
Recombinant subunit vaccine expressed in Pichia pastoris.[3]
On February 21, 2020, the FDA approved Eptinezumab, a
CGRP antibody from Danish pharmaceutical company Lundbeck, for market release
under the brand name Vyepti, used for migraine prevention. Eptinezumab is
expressed in Pichia pastoris cells using recombinant DNA technology and is the
first monoclonal antibody drug expressed in yeast. This work has opened up a
new blue ocean for yeast application in antibody production and cost control in
antibody drug manufacturing.
In the context of intense competition among
pharmaceutical companies, severe product homogenization, and national drug bulk
procurement, pharmaceutical companies face challenges in improving drug
development success rates, reducing production costs, and enhancing core
competitiveness. With the improvement and maturation of the Pichia pastoris
expression system, its characteristics such as low production costs, short
fermentation cycles, ease of cultivation, simple and convenient operation,
capability for large-scale production, genetic stability, and
post-translational protein modifications have made it increasingly favored by
research institutions and pharmaceutical manufacturing companies.
✦ Company Introduction
LKtime Biotech has established recombinant protein expression platforms for Pichia pastoris and Saccharomyces cerevisiae, capable of providing full-process development services from Gene to IND, as well as early clinical sample preparation, with a development cycle of 9-15 months. The company has built a mature 500L microbial fermentation and purification production line that meets GMP requirements, capable of providing drug substance preparation at scales of 10L-50L and 500L.
References
[1] Yeasts as Biopharmaceutical Production Platforms. Front Fungal Biol. 2021.
[2] The humanization of N-glycosylation pathways in yeast. Nat Rev microbiol. 2005; 3(2): 119-28.
[3] Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol. 2020; 235(9): 5867-5881.
